Dissertation
Dissertation > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Livestock > Cow

Molecular Characterization, Expression, and Promoter Methylation Status of the Bovine Synaptonemal Complex Protein 3 Gene

Author ZhuXiang
Tutor LiQiFa
School Nanjing Agricultural College
Course Animal Genetic Breeding and Reproduction
Keywords cattle yak cattle-yak bSYCP3 gene expression methylation male-sterilize
CLC S823
Type Master's thesis
Year 2011
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Synaptonemal complex protein 3 (SYCP3) is a kind of DNA binding protein, located on the lateral elements synaptonemal complex(SC), it makes great contribution to the synapsis of homogenous chromosome and it is necessary on the process of meiosis of male mammals within spermatogenesis. Compare to previous finding in our laboratory, the deletion or low expression of SYCP3 gene induce the same spermatogenesis obstacle phenotype is congruent to the phenotype of cattle-yak, which is male-sterile and meiosis arrest. In order to study of the function of SYCP3 gene and its connection with infertility of male cattle-yak, our study adopt PCR amplifying and cloning sequences to acquire the SYCP3 gene sequence of cattle and yak, using bioinformatics method to analyze the tissue expression characteristic, and the bSYCP3 gene expression levels in testis of cattle, yak and cattle-yak separately; we also exemplify the 5’ultra none-translation region sequence of SYCP3 gene (both cattle and yak), and employ bisulfite sequence technique to explore the difference methylation statute among promoter region of cattle, yak and cattle-yak SYCP3 gene. Results as below:1. bSYCP3 cloning, sequence analysis and evolutionary studies in cattle and yakOur study using the PCR amplifying and cloning sequence technique to get the SYCP3 gene sequence of cattle and yak, and named bSYCP3. The intact SYCP3 gene translation region is 678bp, by comparing the result sequences, their homology value reaches 100%, and get a comparatively high level with other mammals(71%-83%). bSYCP3 gene including 8 exons and 7 introns, and its start code locate in the second exon; the bSYCP3 gene is located in the fifth chromosome. The bSYCP3 gene translates a protein with 225 amino acids resides and by analyzing the amino acid sequences, the bSYCP3 protein contains a Corl motif and two coiled-coil-formings, and its amino acid sequence of Corl motif has a high homology value with other mammals(59%-77%), and been affected by negative selection. The results form phylogenetic analysis (NJ tree), which is built based on amino acid sequence of bSYCP3 and Corl motif, are according to the classic category of animals, which shows that the evolution of mammal SYCP3 gene is conservative, and we can infer it has a large possibility that the SYCP3 protein of cattle and yak have the similar biological function with SYCP3 gene of other mammals, indeed, bSYCP3 gene participates in the process of meiosis and spermatogenesis of male bovine.2. bSYCP3 gene expression analysis in cattle, yak and cattle-yak Our study adopting RT-PCR to make analyze about a series tissue, such as hypothalamus and testis, through the study of multiple organize expression pattern about SYCP3 gene, which is specify expression on bovine testis and comply to the research outcome of other advanced mammals, such as mouse and rat. Meanwhile, the real-time PCR reveals that, the testis bSYCP3 gene expression level of male sterilize in cattle-yak is significant less that the level in cattle and yak (P<0.01), and there is no significant differences between cattle and yak (P>0.05).The phenotypes of meiotic arrest in cattle-yak are nearly the same with phenotypes of male fertility in human and mouse with SYCP3 gene missing or expression reduced. Therefore, we inferred that bSYCP3 gene plays an important role during cattle spermatogenesis and meiosis, and have a certain relationship with male sterility in cattle-yak, which suggest that the low expression level of the gene has close relationship with the male-sterilize.3. bSYCP3 gene promoter methylation analysis in yak and cattle-yakFurther more, we amplify and get 2271bp of the 5’ultral sequence of bSYCP3 gene, then discover a CpG island, which span the promoter region, the first exon and the first intron. Translation binding factor location analyze demonstrated that the core promoter region exist in sensitive methylation sites, such as SP1, which implies that DNA methylation posses the function to modify the expression of bSYCP3 gene. We analyzed the methylation rate of 19 CpG sites in CpG island of bSYCP3 gene form yak and cattle-yak’s testis, finding that the nucleotide sites take up 81.58%(155/190) on the whole sites of cattle testis, and the cattle-yak testis is 86.84%(165/190) actually there is no significant difference between them(P>0.05), even though the methylation level of cattle-yak is high than cattle. In the analyzed methylation situation of the 7th、13th and 17th CpG sites, there is significant difference between them (P<0.05), the level of methylated site in cattle is 0 (0/30), however the level of cattle-yak is 40%(12/20). In short, the research results of the high methylation level of bSYCP3 gene in cattle-yak is in rapport with the low expression level of mRNA and male-sterile, which speculated that the gene methylation may play an important role in modification of bSYCP3 gene mRNA expression, more importantly, have a certain relationship with the meiosis process and male-sterilize of cattle-yak.

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